- Email: [email protected]
Immunity to the liver stage of malaria
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Immunity to the Liver Stage of Malaria A. Suhrbier The search for subunit vaccines against malaria has concentrated on asexual and sexual blood stage and sporozoite antigens. In recent years the search for the basis of the protection against sporozoite challenge obtained in mice immunized with irradiated sporozoitefl has focused attention on the liver or exoerythrocytic (E E ) stage of the malaria life cycle. Here, Andreas Suhrbier looks at the various immune responses that appear to be active against this stage, which was once thought to be immunologically insignificant. The liver stage of malaria has thus emerged as a legitimate target for vaccine development. Mouse and rat malaria models have revealed a large variety of immune mechanisms active against the EE stage of malaria (Fig. 1). Unfortunately these model systems represent unnatural host-parasite combinations and the protective mechanisms appear to differ for each model. Each malaria species has probably evolved to evade the immune responses of its particular host, and whether the following immune responses represent mechanisms relevant to vaccine design for humans has still to be established. Cytotoxic CD8 + T cells Cytotoxic CD8- T lymphocytes (CTL) recognize proteolytically processed fragments of cytoplasmic antigens associated with products of the major histocompatibility complex (MHC) class I (Ref. 2). These immune effector cells have been shown to be the principle protective agent in some strains of mice immunized with irradiated sporozoites 3'4. Hepatocytes have therefore been implicated as the target of protective CTL, since only the EE stage of the malaria life cycle resides within a cell that bears the functional MHC class I antigens required for CTL activity. (Sporozoites attenuated by irradiation are fully capable of invading hepatocytes but the resulting EE parasites grow slowly and perish during the same period in which normal EE parasites reach maturityS.) CTL from mice immunized with irradiated sporozoites kill EE parasites cultured in mouse hepatocytes and appear to be involved in the destruction of EE parasites in the livers of immunized mice 6. A proteolytically processed fragment, or epitope, derived from one region of the circumsporozoite protein (CSP) has been shown to be a target for Andreas Suhrb~er~sat the Queensland Institute of Medical Research. Bramston Terrace. Brisbane, Queensland. Australia 4006. and Imperial College. Department of B~ology,Pnnce Consort Rd. London SW7 4BB, UK
CTL 7'8. Some CTL clones specific for this epitope also protect mice against sporozoite challenge if administered in high numbers with interleukin 2 (IL-2) (Ref. 9). In addition, oral-attenuated Salmonella typhimurium recombinants containing the Plasmodium berghei CSP gene protect mice by induction of specific CD8 + T cells. By residing within macrophages these bacteria presumably deliver the CSP to the required intracellular location, where it can be processed and presented in association with MHC class I (Ref. 10). The P. falciparum CSP CTL epitope has recently been defined ~~,~z and overlaps the region identified in mouse experiments. Unfortunately, this epitope lies within a region of the CSP that varies considerably between different isolates ~3. The extent of such variation across the globe and therefore the number of variant sequences that would have to be included in subunit vaccines is unknown. Conceivably, a small number of epitopes might be engineered to induce crossreactive CTL capable of recognizing most or all the variants. Alternatively, region-specific vaccines might be envisaged, which contain only those variants present within that region ~4. Experiments in mice have shown that certain strains are incapable of making a CD4 ~ T-cell response to the CSP. Similar immune restriction may also prevent an unknown percentage of the human population from raising a CD8 ÷ T-cell response to these epitopes ~s. Before we rush headlong down this path, it is important to remember that all the protection experiments described above were performed in mice principally using P. berghei as well as P. yoelii, whose natural vertebrate hosts are not mice but Thamnomys surdaster and T. rutilans, respectively ~6. Immunization with irradiated sporozoites, using correctly matched host-parasite combinations including P. falciparum in humans, is unfortunately not as effective as using P. berghei in mice ]'17. In addition, effective irradiated sporozoite immunity is not obtained in all mouse strain-P, berghei or P. yoelii combinations, nor do all the combinations depend on CD8 + CTL for protection ~s. Although immune response and other background genes play an important part in controlling these responses, there also appears to be a correlation between infectivity and protection. Individual mouse strain-P, berghei or P. yoelii combinations, where nonirradiated sporozoites are highly infective to the mouse strain ~9, are hard to protect with irradiated sporozoites of the same species and develop CTL-independent protection. Conversely, combinations with poor infectivity are
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IFN ,~
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=" Sporozoites
~-
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cell
Antibody
Phagocytosis
t
/
Merozoites •
•
• • o
i[E:;rtaS/~e
CRP
Kupffercell Antibody
CD4+ T cells ? Fig. I. Immune responses to the liver stageof malaria. Crisis-reactive protein (CRP) is synthesized by the hepatocyte in response to interleukin 6. Nitrogen oxides (NO) are generated by Kupffer cells in response to interleukin 6. Interleukin I and turnout necrosis factor are believed to induce nonporenchymal liver cells to produce interleukin 6. Exoerythroc~c (EE); gamma interferon (IFN-y); cytotoxic T lymphocytes (CTL).
easy to protect and develop CTL-dependent protection. A possible explanation for the latter correlation is offered by the rather artifactual behaviour of P. berghei sporozoites. Shortly after invasion of the hepatocyte, in vitro, large numbers of these sporozoites (whether irradiated or not) perish, shedding sporozoite antigens (notably the CSP) into the cytoplasm of the hepatocyte, where they can be processed and associate with MHC class I (Ref. 5). This behaviour may explain the low infectivity of these sporozoites to unnatural host hepatocytes x9 and would have the effect, in viva, of (1) inducing large numbers of sporozoite-specific CTL during immunization and (2) recruiting these CTL to the liver and activating them after sporozoite challenge. These observations beg the question of which of these mouse models describe immune responses that also operate in humans. For instance, poor infectivity is not thought to be a feature of human malarias 2°. It remains to be established whether human EE parasites in human hepatocytes are targets for protective CTL and whether a vaccine can be designed to induce both (1) and (2) above 1°. CD4 + T cells Recently, CD4 ~ T cells have been shown to mediate protection against sporozoite challenge. A CD4 ÷ T-cell clone, which has cytotoxic activity and secretes gamma interferon (IFN-y) and IL-2, was able to confer protection against challenge with P. berghei sporozoites2t. CSP-specific CD4 ÷ T-cell clones synthesizing IFN and interleukins 2, 4, 5 and 6 were also shown to protect against P. yoelii sporozoites22. These cells might act (1) via IFN, (2) via IL-6, (3) by cytotoxic activity against infected
hepatocytes through IFN-induced MHC class II (not normally expressed by hepatocytes) and/or (4) through Kupffer cells. Phagocytic cells are probably required for the generation of CD4 ÷ T cells during irradiated sporozoite immunization, as they can present sporozoite/ EE antigens via the required MHC class II. CD4 ÷ T cells may also be required to give help for the generation of CD8 + T cells. Liver-stage antigens The CSP may not be the primary target of protective mechanisms induced by irradiated sporozoites. There is good circumstantial evidence that non-CSP antigens synthesized by the EE parasites derived from irradiated sporozoites are required to induce protection s'23 and direct evidence for this has recently been obtained by Hollingdale and others z4. Immunization with a synthetic peptide epitope containing a sequence from a P. berghei liver-specific protein, LSA 2, protects mice against sporozoite challenge by eliciting CTL capable of killing cultured EE parasites. This is the first report of a successful subunit EE stage vaccine. Liver stages share large numbers of antigens with both the sporozoite and blood stages2~as well as synthesizing antigens specific to the liver stage 24'26'27. Many more targets of protective immunity are likely to emerge and it is hoped that some of these will not suffer from the problems of variation and nonresponsiveness associated with the CSP. Cytokines IFN, IL- 1, IL-6 and tumour necrosis factor (TNF) have all been shown to inhibit the development of
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EE parasites 28. IFN appears to be able to directly inhibit intracellular development of the EE parasite in cultured liver cellsz9 and will protect against sporozoite challenge if administered systemically, in vivo 3°. Anti-IFN antibody administered to mice immunized with irradiated sporozoites also reduces the level of protection to sporozoite challenge4. The source of IFN in P. berghei-immunized mice is believed to be active CTL in the liver, where a local paracrine effect may contribute to protection 31. This effect would not require that all EE parasites be killed directly by CTL; growth of neighbouring parasites might be inhibited by IFN secreted by the few CTL that do encounter EE parasites. However, a challenge with a small number of sporozoites (as is believed to occur in human malaria 2°) may induce insufficient local IFN to protect the host. P. berghei sporozoites for laboratory use are usually derived from very heavily infected mosquito salivary glands, which are dissected and disrupted to release the sporozoites. A high percentage of these sporozoites appear not to be viable and, possibly as a result, the majority of intravenously injected sporozoites localize to the spleen 3z. Spleen cells (probably CD4 ÷ T cells activated by macrophages) have been shown to synthesize IFN in response to sporozoite inoculation33 and may produce sufficient systemic IFN to protect the host. However, in model systems dependant on CD8 ÷ T cells for protection, depletion of CD4 ÷ T cells does not effect immunity. This might suggest, although it has not been directly shown, that spleen-derived IFN does not contribute to protection in these models. The importance of IFN in model systems with higher sporozoite viability (and which do not rely on CTL for protection) should be tested since the large-scale delivery of inoculated sporozoites into the spleen is not thought to occur under natural conditions in human malaria, where most of the sporozoites probably end up in the hepatocyte ~. IFN might operate not only by direct activity on hepatocytes but also by activating phagocytic cells (eg. Kupffer cells). In either event, the induction of high levels of IFN by vaccination carries the risk of inducing potentially hazardous pathology 34. The anti-EE-stage activity of IL-1 may in part be mediated via crisis-reactive protein (CRP) and other inflammatory factors. IL-1 stimulates hepatocytes, via IL-6, to synthesize CRP, which has been shown to bind sporozoites and inhibit the early phase of EE infection. TNF, probably derived from macrophages, and IL-1 stimulate nonparenchymal liver cells to synthesize IL-6, which may then result in Kupffer cells producing nitrogen oxides from arginine. Nitrogen oxides are thought to reduce the ATP level in neighbouring hepatocytes thus inhibiting EE development2s.
Phagocytic cells The phagocytic cells of the liver, the Kupffer cells, are not thought to destroy significant numbers of
Pares~to!ogy Today. vol 7. no 7. 1991
sporozoites in nonimmune mice 35. In fact, based on electronmicroscopic studies of P. berghei in rats, it has been proposed that sporozoites travel through these cells to gain access to the hepatocytes36. However, in the presence of anti-sporozoite antibodies or IFN, sporozoites are likely to be destroyed by the Kupffer cells 3s. Such activity may directly contribute to protection and might also result in activation of CD4 ÷ and, directly/indirectly, CD8 + T cells 1'1°. The relative importance of phagocytes in the spleen and liver in generating protective responses is not clear. Kupffer cells also phagocytose many of the emerging EE stage merozoites in rats 37and in humans 3s. In the latter case, infiltrating macrophages and neutrophils also appear to be active. These phagocytic activities are likely to be enhanced by (1) antibodies specific for EE antigens, which surround the EE merozoites as they are released from the mature EE parasite, (2) antibodies directed at merozoite antigens and (3) IFN. A malaria heat shock protein has been found to be expressed on cultured EE stages, and up to 50% of parasites are destroyed when anti-heat shock protein antibody and nonparenchymal liver cells (principally Kupffer cells) are added 39. In rats inoculated with large numbers of P. berghei sporozoites, approximately 30% of nearly mature EE parasites degenerate and are surrounded by a cellular infiltrate of Kupffer cells, monocytes/macrophages and neutrophils4°. However, inflammatory infiltrates around EE parasites are very rare events in human malarias 3s. The size and number of EE parasites are invariably larger when they reside in their natural host cells, probably because hepatocytes from rats and particularly mice 19 provide suboptimal conditions for parasite growth. The activity of phagocytes against EE parasites may be due to degenerating or fragile EE parasites growing in suboptimal host cells41.
The future Mouse models have revealed a plethora of immune responses capable of destroying EE parasites (Fig. 1). The human parasites may behave very differently to these models and which of these mechanisms, if any, operate in humans will be a subject of controversy. However, vaccines may not need to mimic natural immune mechanisms, provided they induce protective responses. Only experiments using human material and human-specific Plasmodium and, finally, human trials are eventually likely to define a suitable vaccine. As our knowledge of the immune responses against malaria has developed, so has our understanding of how the malaria parasite has evolved to evade them 42. In several years' time we may, for instance, read how the EE parasite of P. falciparum specifically inhibits the peptide transporters 43, which would normally deliver CTL epitopes from the parasite into the cytoplasm of the infected hepatocyte.
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The plasmodial parasites have been studying vertebrate immune systems for millions of years; we have only just begun.
Acknowledgements The author would hke to tl-,an~ Michael Good, Tom Burkot and Annette Fernan for their help in writing this article. References 1 Nussenzweig,V. andNussenzweig, R.S.(1989)Adv. lmmunol. 45,
283-334 2 Townsend, A.R. and Bodmer, H. (1989)Annu. Rev. lmmunol. 7, 601-625 3 Weiss, W.R. etal. (1988)Proc. NatlAcad. Sci. USA 85,573-576 4 Schofield,L. et al. (1987)Nature 330,664-666 5 Suhrbier, A. et al. (1990) Infect. lmmun. 58, 2834-2839 6 Hoffman, S.L. etal. (1989)Science244, 1078-1081 7 Kumar, S. etal. (1988)Nature334,258-260 8 Weiss, W.R. etal. (1990)ff. Exp. Med. 171,763-773 9 Romero, P. et al. (1989)Nature 341,323-326 10 Aggarwal,A. etal. (1990)ff. Exp. Med. 172, 1083-1090 11 Doolan, D., Houghton, R.A. and Good, M.F. Int. lmmunol. (in press) 12 Hoffman, S.L. et al. (1991) Proc. NatlAcad. Sci. USA 88, 3300-3304 13 Lockyer, M.J. et al. (1990) M ol. Bioc hem . P arasitol. 37,275-280 14 Nussenzweig,R.S. and Yoshida, N. (1990)Immunol. Lett. 25, 21-22 15 Good,M.F. etal. (1989)Blood74, 895-900 16 KiUick-Kendrick,R. (1978)inRodentMalaria(Killick-Kendrick, R. and Peters, W., eds), pp 1-52, AcademicPress
17 Druilhe, P. (1989)in New Strategies in Parasitology (McAdam, J. K. P. H., ed. ), pp 39-48, Churchill-Livingstone 18 Weiss, W.R. (1990)lmmunol. Lett. 25, 39-42 19 Coosemans,M. et al. (1981)Ann. Sac. Belg. Med. Trap. 61,349-368 20 Rosenberg, R. et al. (1990) Trans. R. Sac. Trap. Med. Hyg. 84, 209-212 21 Tsuji, M. etal. (1990)J. Exp. Med. 172, 1353-1357 22 Del Giudice, G. et al. (1990) Immunol. Lett. 25, 59-64 23 Mellouk, S. et al. (1990) Lancet 335, 721 24 Hollingdale, M.R. etal. (1990)Immunol. Lett. 25, 71-76 25 Szarfman,A. et al. (1988) Parasite Immunol. 10,339-351 26 Guerin-Marchand, C. etal. (1987)Nature 329, 164-167 27 Suhrbier,A. etal. (1990) Parasite lmmunol. 12,473-481 28 Mazier D. etaL (1990)Immunol. Len. 25, 65-70 29 Ferreira, A. etal. (1986)Science 232,881-884 30 Maheshwari,R.K. etal. (1990) Immunol. Left. 25, 53-58 31 Schofield,L. (1989) Exp. Parasitol. 68,357-364 32 Ferreira, A. et al. (1986)Mol. Biochem. Parasitol. 19,103-109 33 Ojo-Amaize,E.A. etal. (1984)J. Immunol. 133, 1005-1009 34 Playfair, J.H.L. etal. (1990)Immunol. Today 11,25-27 35 Seguin, M.C., Ballou,W.R. and Nacy, C.A. (1989)J. Immunol. 143, 1716-1722 36 Meis, J.F.G.M. etal. (1983) Parasitology 86, 231-242 37 Terzakis, J.A. etal. (1979)J. Protozool. 26,385-389 38 Garnham, P.C.C. and Bray, R.S. (1956)Rev. Bras. Malartol. Doecnas Trop. 8,152-160 39 Renia, L. et al. (1990) Eur. J. Imrnunol. 20, 1445-1449 40 Meis, J.F.G.M. etal. (1987)Am.J. Trop.Med. Hyg. 37,506-510 41 Jap, P.H.K. etal. (1982)Parasitology 85,263-269 42 Good, M.F., Kumar, S. and Miller, L.H. (1988)Immunol. Today 9, 351-355 43 Spies, T. et al. (1990) Nature 348,744-747
The Interaction Between Intestinal Mucus Glycoproteins and Enteric Infections S-K. Tse and K. Chadee Adherence of pathogenic enteric organisms w specific receptors on mucosal surfaces is widely recognized as an important first step in the initiation of infectious diseases. The specific interactions whereby parasites and bacteria exploit mucus substrates for colonization, and the host uses them as a nonimmunological defense mechanism, is only now being unravelled. In this review, S il-King Tse and Kris Chadee discuss various hypothetical models for interaction, including the role of the immune system in the regulation of mucus secretion. Pathogens can attach directly to mucus gel receptors or to specific receptors on enterocytes. Many motile bacteria respond to epithelial cell chemotactic factors (taxins) or have proteinase and glycosidase (mucinase) activity that enable them to cross the thick viscous layer of mucus to attach to or invade epithelial cells. The attachment process of bacteria to mucus substrates is initiated by fimbriae or pili (adhesins) which have lectin activity. Protozoan parasites have lectin molecules that enable them to Sii-King Tse and Krls Chadee are at the InstLtute of Parasitology of McGII University. Macdonald CampLs, 21 I I I Lakeshore Road. SteAnne-de-Bellevue, Quebec. Canada H9X I C0. ~ ) 1991 ~-se,ie' Sceece P~b,,she,~ ! d ,,~Ki~169. 4'S, 1 9 1 ~ 0 2 ~
attach not only to mucus substrates but also to enterocytes. Mucus receptors have not been identified in helminths and do not elicit mucus secretion in the absence of an immune response. The advantage of having mucus receptors for enteric pathogens is that they can (1) trap and remove potentially harmful organisms, (2) enable beneficial microorganisms to colonize and (3) facilitate competitive colonization by nonpathogens, preventing subsequent colonization by pathogens and thus invasion. Bacteria as well as eukaryotic parasites use the sugar moiety of mucus glycoproteins as receptors for attachment. Different mucin species expressing differences in sugar composition and polypeptide backbone have implications in health and disease and an intact mucin molecule is necessary for execution of its primary function as the first line of host defense against enteric pathogens. M u c u s structure and function
Intestinal mucus is secreted by epithelial goblet ceils by compound exocytosis, the fusion of apical mucus granules with the luminal plasma membrane resulting in release of mucus granules ~. It forms the viscous gel-like layer lining the gut and consists of a
Poros~[o!ogy Todey. vol 7, no 7. 19g i
Immunity to the Liver Stage of Malaria A. Suhrbier The search for subunit vaccines against malaria has concentrated on asexual and sexual blood stage and sporozoite antigens. In recent years the search for the basis of the protection against sporozoite challenge obtained in mice immunized with irradiated sporozoitefl has focused attention on the liver or exoerythrocytic (E E ) stage of the malaria life cycle. Here, Andreas Suhrbier looks at the various immune responses that appear to be active against this stage, which was once thought to be immunologically insignificant. The liver stage of malaria has thus emerged as a legitimate target for vaccine development. Mouse and rat malaria models have revealed a large variety of immune mechanisms active against the EE stage of malaria (Fig. 1). Unfortunately these model systems represent unnatural host-parasite combinations and the protective mechanisms appear to differ for each model. Each malaria species has probably evolved to evade the immune responses of its particular host, and whether the following immune responses represent mechanisms relevant to vaccine design for humans has still to be established. Cytotoxic CD8 + T cells Cytotoxic CD8- T lymphocytes (CTL) recognize proteolytically processed fragments of cytoplasmic antigens associated with products of the major histocompatibility complex (MHC) class I (Ref. 2). These immune effector cells have been shown to be the principle protective agent in some strains of mice immunized with irradiated sporozoites 3'4. Hepatocytes have therefore been implicated as the target of protective CTL, since only the EE stage of the malaria life cycle resides within a cell that bears the functional MHC class I antigens required for CTL activity. (Sporozoites attenuated by irradiation are fully capable of invading hepatocytes but the resulting EE parasites grow slowly and perish during the same period in which normal EE parasites reach maturityS.) CTL from mice immunized with irradiated sporozoites kill EE parasites cultured in mouse hepatocytes and appear to be involved in the destruction of EE parasites in the livers of immunized mice 6. A proteolytically processed fragment, or epitope, derived from one region of the circumsporozoite protein (CSP) has been shown to be a target for Andreas Suhrb~er~sat the Queensland Institute of Medical Research. Bramston Terrace. Brisbane, Queensland. Australia 4006. and Imperial College. Department of B~ology,Pnnce Consort Rd. London SW7 4BB, UK
CTL 7'8. Some CTL clones specific for this epitope also protect mice against sporozoite challenge if administered in high numbers with interleukin 2 (IL-2) (Ref. 9). In addition, oral-attenuated Salmonella typhimurium recombinants containing the Plasmodium berghei CSP gene protect mice by induction of specific CD8 + T cells. By residing within macrophages these bacteria presumably deliver the CSP to the required intracellular location, where it can be processed and presented in association with MHC class I (Ref. 10). The P. falciparum CSP CTL epitope has recently been defined ~~,~z and overlaps the region identified in mouse experiments. Unfortunately, this epitope lies within a region of the CSP that varies considerably between different isolates ~3. The extent of such variation across the globe and therefore the number of variant sequences that would have to be included in subunit vaccines is unknown. Conceivably, a small number of epitopes might be engineered to induce crossreactive CTL capable of recognizing most or all the variants. Alternatively, region-specific vaccines might be envisaged, which contain only those variants present within that region ~4. Experiments in mice have shown that certain strains are incapable of making a CD4 ~ T-cell response to the CSP. Similar immune restriction may also prevent an unknown percentage of the human population from raising a CD8 ÷ T-cell response to these epitopes ~s. Before we rush headlong down this path, it is important to remember that all the protection experiments described above were performed in mice principally using P. berghei as well as P. yoelii, whose natural vertebrate hosts are not mice but Thamnomys surdaster and T. rutilans, respectively ~6. Immunization with irradiated sporozoites, using correctly matched host-parasite combinations including P. falciparum in humans, is unfortunately not as effective as using P. berghei in mice ]'17. In addition, effective irradiated sporozoite immunity is not obtained in all mouse strain-P, berghei or P. yoelii combinations, nor do all the combinations depend on CD8 + CTL for protection ~s. Although immune response and other background genes play an important part in controlling these responses, there also appears to be a correlation between infectivity and protection. Individual mouse strain-P, berghei or P. yoelii combinations, where nonirradiated sporozoites are highly infective to the mouse strain ~9, are hard to protect with irradiated sporozoites of the same species and develop CTL-independent protection. Conversely, combinations with poor infectivity are
161
Paros~'.oiogy Today. voL 7, no 7, i991
IFN ,~
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=" Sporozoites
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cell
Antibody
Phagocytosis
t
/
Merozoites •
•
• • o
i[E:;rtaS/~e
CRP
Kupffercell Antibody
CD4+ T cells ? Fig. I. Immune responses to the liver stageof malaria. Crisis-reactive protein (CRP) is synthesized by the hepatocyte in response to interleukin 6. Nitrogen oxides (NO) are generated by Kupffer cells in response to interleukin 6. Interleukin I and turnout necrosis factor are believed to induce nonporenchymal liver cells to produce interleukin 6. Exoerythroc~c (EE); gamma interferon (IFN-y); cytotoxic T lymphocytes (CTL).
easy to protect and develop CTL-dependent protection. A possible explanation for the latter correlation is offered by the rather artifactual behaviour of P. berghei sporozoites. Shortly after invasion of the hepatocyte, in vitro, large numbers of these sporozoites (whether irradiated or not) perish, shedding sporozoite antigens (notably the CSP) into the cytoplasm of the hepatocyte, where they can be processed and associate with MHC class I (Ref. 5). This behaviour may explain the low infectivity of these sporozoites to unnatural host hepatocytes x9 and would have the effect, in viva, of (1) inducing large numbers of sporozoite-specific CTL during immunization and (2) recruiting these CTL to the liver and activating them after sporozoite challenge. These observations beg the question of which of these mouse models describe immune responses that also operate in humans. For instance, poor infectivity is not thought to be a feature of human malarias 2°. It remains to be established whether human EE parasites in human hepatocytes are targets for protective CTL and whether a vaccine can be designed to induce both (1) and (2) above 1°. CD4 + T cells Recently, CD4 ~ T cells have been shown to mediate protection against sporozoite challenge. A CD4 ÷ T-cell clone, which has cytotoxic activity and secretes gamma interferon (IFN-y) and IL-2, was able to confer protection against challenge with P. berghei sporozoites2t. CSP-specific CD4 ÷ T-cell clones synthesizing IFN and interleukins 2, 4, 5 and 6 were also shown to protect against P. yoelii sporozoites22. These cells might act (1) via IFN, (2) via IL-6, (3) by cytotoxic activity against infected
hepatocytes through IFN-induced MHC class II (not normally expressed by hepatocytes) and/or (4) through Kupffer cells. Phagocytic cells are probably required for the generation of CD4 ÷ T cells during irradiated sporozoite immunization, as they can present sporozoite/ EE antigens via the required MHC class II. CD4 ÷ T cells may also be required to give help for the generation of CD8 + T cells. Liver-stage antigens The CSP may not be the primary target of protective mechanisms induced by irradiated sporozoites. There is good circumstantial evidence that non-CSP antigens synthesized by the EE parasites derived from irradiated sporozoites are required to induce protection s'23 and direct evidence for this has recently been obtained by Hollingdale and others z4. Immunization with a synthetic peptide epitope containing a sequence from a P. berghei liver-specific protein, LSA 2, protects mice against sporozoite challenge by eliciting CTL capable of killing cultured EE parasites. This is the first report of a successful subunit EE stage vaccine. Liver stages share large numbers of antigens with both the sporozoite and blood stages2~as well as synthesizing antigens specific to the liver stage 24'26'27. Many more targets of protective immunity are likely to emerge and it is hoped that some of these will not suffer from the problems of variation and nonresponsiveness associated with the CSP. Cytokines IFN, IL- 1, IL-6 and tumour necrosis factor (TNF) have all been shown to inhibit the development of
162_
EE parasites 28. IFN appears to be able to directly inhibit intracellular development of the EE parasite in cultured liver cellsz9 and will protect against sporozoite challenge if administered systemically, in vivo 3°. Anti-IFN antibody administered to mice immunized with irradiated sporozoites also reduces the level of protection to sporozoite challenge4. The source of IFN in P. berghei-immunized mice is believed to be active CTL in the liver, where a local paracrine effect may contribute to protection 31. This effect would not require that all EE parasites be killed directly by CTL; growth of neighbouring parasites might be inhibited by IFN secreted by the few CTL that do encounter EE parasites. However, a challenge with a small number of sporozoites (as is believed to occur in human malaria 2°) may induce insufficient local IFN to protect the host. P. berghei sporozoites for laboratory use are usually derived from very heavily infected mosquito salivary glands, which are dissected and disrupted to release the sporozoites. A high percentage of these sporozoites appear not to be viable and, possibly as a result, the majority of intravenously injected sporozoites localize to the spleen 3z. Spleen cells (probably CD4 ÷ T cells activated by macrophages) have been shown to synthesize IFN in response to sporozoite inoculation33 and may produce sufficient systemic IFN to protect the host. However, in model systems dependant on CD8 ÷ T cells for protection, depletion of CD4 ÷ T cells does not effect immunity. This might suggest, although it has not been directly shown, that spleen-derived IFN does not contribute to protection in these models. The importance of IFN in model systems with higher sporozoite viability (and which do not rely on CTL for protection) should be tested since the large-scale delivery of inoculated sporozoites into the spleen is not thought to occur under natural conditions in human malaria, where most of the sporozoites probably end up in the hepatocyte ~. IFN might operate not only by direct activity on hepatocytes but also by activating phagocytic cells (eg. Kupffer cells). In either event, the induction of high levels of IFN by vaccination carries the risk of inducing potentially hazardous pathology 34. The anti-EE-stage activity of IL-1 may in part be mediated via crisis-reactive protein (CRP) and other inflammatory factors. IL-1 stimulates hepatocytes, via IL-6, to synthesize CRP, which has been shown to bind sporozoites and inhibit the early phase of EE infection. TNF, probably derived from macrophages, and IL-1 stimulate nonparenchymal liver cells to synthesize IL-6, which may then result in Kupffer cells producing nitrogen oxides from arginine. Nitrogen oxides are thought to reduce the ATP level in neighbouring hepatocytes thus inhibiting EE development2s.
Phagocytic cells The phagocytic cells of the liver, the Kupffer cells, are not thought to destroy significant numbers of
Pares~to!ogy Today. vol 7. no 7. 1991
sporozoites in nonimmune mice 35. In fact, based on electronmicroscopic studies of P. berghei in rats, it has been proposed that sporozoites travel through these cells to gain access to the hepatocytes36. However, in the presence of anti-sporozoite antibodies or IFN, sporozoites are likely to be destroyed by the Kupffer cells 3s. Such activity may directly contribute to protection and might also result in activation of CD4 ÷ and, directly/indirectly, CD8 + T cells 1'1°. The relative importance of phagocytes in the spleen and liver in generating protective responses is not clear. Kupffer cells also phagocytose many of the emerging EE stage merozoites in rats 37and in humans 3s. In the latter case, infiltrating macrophages and neutrophils also appear to be active. These phagocytic activities are likely to be enhanced by (1) antibodies specific for EE antigens, which surround the EE merozoites as they are released from the mature EE parasite, (2) antibodies directed at merozoite antigens and (3) IFN. A malaria heat shock protein has been found to be expressed on cultured EE stages, and up to 50% of parasites are destroyed when anti-heat shock protein antibody and nonparenchymal liver cells (principally Kupffer cells) are added 39. In rats inoculated with large numbers of P. berghei sporozoites, approximately 30% of nearly mature EE parasites degenerate and are surrounded by a cellular infiltrate of Kupffer cells, monocytes/macrophages and neutrophils4°. However, inflammatory infiltrates around EE parasites are very rare events in human malarias 3s. The size and number of EE parasites are invariably larger when they reside in their natural host cells, probably because hepatocytes from rats and particularly mice 19 provide suboptimal conditions for parasite growth. The activity of phagocytes against EE parasites may be due to degenerating or fragile EE parasites growing in suboptimal host cells41.
The future Mouse models have revealed a plethora of immune responses capable of destroying EE parasites (Fig. 1). The human parasites may behave very differently to these models and which of these mechanisms, if any, operate in humans will be a subject of controversy. However, vaccines may not need to mimic natural immune mechanisms, provided they induce protective responses. Only experiments using human material and human-specific Plasmodium and, finally, human trials are eventually likely to define a suitable vaccine. As our knowledge of the immune responses against malaria has developed, so has our understanding of how the malaria parasite has evolved to evade them 42. In several years' time we may, for instance, read how the EE parasite of P. falciparum specifically inhibits the peptide transporters 43, which would normally deliver CTL epitopes from the parasite into the cytoplasm of the infected hepatocyte.
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The plasmodial parasites have been studying vertebrate immune systems for millions of years; we have only just begun.
Acknowledgements The author would hke to tl-,an~ Michael Good, Tom Burkot and Annette Fernan for their help in writing this article. References 1 Nussenzweig,V. andNussenzweig, R.S.(1989)Adv. lmmunol. 45,
283-334 2 Townsend, A.R. and Bodmer, H. (1989)Annu. Rev. lmmunol. 7, 601-625 3 Weiss, W.R. etal. (1988)Proc. NatlAcad. Sci. USA 85,573-576 4 Schofield,L. et al. (1987)Nature 330,664-666 5 Suhrbier, A. et al. (1990) Infect. lmmun. 58, 2834-2839 6 Hoffman, S.L. etal. (1989)Science244, 1078-1081 7 Kumar, S. etal. (1988)Nature334,258-260 8 Weiss, W.R. etal. (1990)ff. Exp. Med. 171,763-773 9 Romero, P. et al. (1989)Nature 341,323-326 10 Aggarwal,A. etal. (1990)ff. Exp. Med. 172, 1083-1090 11 Doolan, D., Houghton, R.A. and Good, M.F. Int. lmmunol. (in press) 12 Hoffman, S.L. et al. (1991) Proc. NatlAcad. Sci. USA 88, 3300-3304 13 Lockyer, M.J. et al. (1990) M ol. Bioc hem . P arasitol. 37,275-280 14 Nussenzweig,R.S. and Yoshida, N. (1990)Immunol. Lett. 25, 21-22 15 Good,M.F. etal. (1989)Blood74, 895-900 16 KiUick-Kendrick,R. (1978)inRodentMalaria(Killick-Kendrick, R. and Peters, W., eds), pp 1-52, AcademicPress
17 Druilhe, P. (1989)in New Strategies in Parasitology (McAdam, J. K. P. H., ed. ), pp 39-48, Churchill-Livingstone 18 Weiss, W.R. (1990)lmmunol. Lett. 25, 39-42 19 Coosemans,M. et al. (1981)Ann. Sac. Belg. Med. Trap. 61,349-368 20 Rosenberg, R. et al. (1990) Trans. R. Sac. Trap. Med. Hyg. 84, 209-212 21 Tsuji, M. etal. (1990)J. Exp. Med. 172, 1353-1357 22 Del Giudice, G. et al. (1990) Immunol. Lett. 25, 59-64 23 Mellouk, S. et al. (1990) Lancet 335, 721 24 Hollingdale, M.R. etal. (1990)Immunol. Lett. 25, 71-76 25 Szarfman,A. et al. (1988) Parasite Immunol. 10,339-351 26 Guerin-Marchand, C. etal. (1987)Nature 329, 164-167 27 Suhrbier,A. etal. (1990) Parasite lmmunol. 12,473-481 28 Mazier D. etaL (1990)Immunol. Len. 25, 65-70 29 Ferreira, A. etal. (1986)Science 232,881-884 30 Maheshwari,R.K. etal. (1990) Immunol. Left. 25, 53-58 31 Schofield,L. (1989) Exp. Parasitol. 68,357-364 32 Ferreira, A. et al. (1986)Mol. Biochem. Parasitol. 19,103-109 33 Ojo-Amaize,E.A. etal. (1984)J. Immunol. 133, 1005-1009 34 Playfair, J.H.L. etal. (1990)Immunol. Today 11,25-27 35 Seguin, M.C., Ballou,W.R. and Nacy, C.A. (1989)J. Immunol. 143, 1716-1722 36 Meis, J.F.G.M. etal. (1983) Parasitology 86, 231-242 37 Terzakis, J.A. etal. (1979)J. Protozool. 26,385-389 38 Garnham, P.C.C. and Bray, R.S. (1956)Rev. Bras. Malartol. Doecnas Trop. 8,152-160 39 Renia, L. et al. (1990) Eur. J. Imrnunol. 20, 1445-1449 40 Meis, J.F.G.M. etal. (1987)Am.J. Trop.Med. Hyg. 37,506-510 41 Jap, P.H.K. etal. (1982)Parasitology 85,263-269 42 Good, M.F., Kumar, S. and Miller, L.H. (1988)Immunol. Today 9, 351-355 43 Spies, T. et al. (1990) Nature 348,744-747
The Interaction Between Intestinal Mucus Glycoproteins and Enteric Infections S-K. Tse and K. Chadee Adherence of pathogenic enteric organisms w specific receptors on mucosal surfaces is widely recognized as an important first step in the initiation of infectious diseases. The specific interactions whereby parasites and bacteria exploit mucus substrates for colonization, and the host uses them as a nonimmunological defense mechanism, is only now being unravelled. In this review, S il-King Tse and Kris Chadee discuss various hypothetical models for interaction, including the role of the immune system in the regulation of mucus secretion. Pathogens can attach directly to mucus gel receptors or to specific receptors on enterocytes. Many motile bacteria respond to epithelial cell chemotactic factors (taxins) or have proteinase and glycosidase (mucinase) activity that enable them to cross the thick viscous layer of mucus to attach to or invade epithelial cells. The attachment process of bacteria to mucus substrates is initiated by fimbriae or pili (adhesins) which have lectin activity. Protozoan parasites have lectin molecules that enable them to Sii-King Tse and Krls Chadee are at the InstLtute of Parasitology of McGII University. Macdonald CampLs, 21 I I I Lakeshore Road. SteAnne-de-Bellevue, Quebec. Canada H9X I C0. ~ ) 1991 ~-se,ie' Sceece P~b,,she,~ ! d ,,~Ki~169. 4'S, 1 9 1 ~ 0 2 ~
attach not only to mucus substrates but also to enterocytes. Mucus receptors have not been identified in helminths and do not elicit mucus secretion in the absence of an immune response. The advantage of having mucus receptors for enteric pathogens is that they can (1) trap and remove potentially harmful organisms, (2) enable beneficial microorganisms to colonize and (3) facilitate competitive colonization by nonpathogens, preventing subsequent colonization by pathogens and thus invasion. Bacteria as well as eukaryotic parasites use the sugar moiety of mucus glycoproteins as receptors for attachment. Different mucin species expressing differences in sugar composition and polypeptide backbone have implications in health and disease and an intact mucin molecule is necessary for execution of its primary function as the first line of host defense against enteric pathogens. M u c u s structure and function
Intestinal mucus is secreted by epithelial goblet ceils by compound exocytosis, the fusion of apical mucus granules with the luminal plasma membrane resulting in release of mucus granules ~. It forms the viscous gel-like layer lining the gut and consists of a